JP7104399B2 - Photoreactive artificial amino acids and photocrosslinkable artificial polypeptides - Google Patents

Description

The present invention relates to a photoreactive artificial amino acid and a photocrosslinkable artificial polypeptide into which the photoreactive artificial amino acid is introduced.

Basic techniques in the field of molecular biology include nucleic acid ligation and nucleic acid cross-linking. Nucleic acid ligation and cross-linking are used, for example, in combination with hybridization for gene introduction, base sequence detection, or, for example, inhibition of gene expression. To this end, nucleic acid ligation and cross-linking techniques are not limited to basic research in molecular biology, but include, for example, diagnostics and treatments in the medical field, or enzymes in the development and manufacture of therapeutic agents and diagnostic agents, and in the industrial and agricultural fields. It is an extremely important technology used for the development and production of microorganisms.

As a photoreaction technique for nucleic acids, a photolinking technique using 5-cyanovinyldeoxyuridine (Patent Document 1: Patent No. 3753938, Patent Document 2: Patent No. 3753942), modification having a 3-vinylcarbazole structure as a base site. There is a photocrossing technique using nucleoside (Patent Document 3: Patent No. 4814904, Patent Document 4: Patent No. 4940311). Further, there is a photocrosslinking technique using a compound in which the structure of the so-called sugar portion of a modified nucleoside having a 3-vinylcarbazole structure as a base site is replaced with a chain alkylamide (Patent Document 5: Patent No. 5925383).

Japanese Patent No. 3753938 Japanese Patent No. 3753942 Japanese Patent No. 4814904 Japanese Patent No. 4940311 Japanese Patent No. 5925383

With regard to cross-linking between nucleic acids, techniques for forming photocrosslinks are being accumulated as described above. If such photocrosslink formation is also possible for crosslinks between polypeptides and nucleic acids, it will provide an important basic technique in molecular biology.

Therefore, an object of the present invention is to provide a novel photoreactive compound that can be used for the formation of photocrosslinks between nucleic acids and polypeptides.

The present inventor has been diligently researching a photocrossing technique between a polypeptide and a nucleic acid, and introduced a modified amino acid compound (artificial amino acid) having a vinylcarbazole structure as a side chain portion of the amino acid into the polypeptide chain. The present invention was reached by finding that when an artificial polypeptide is produced, the artificial polypeptide can form a photobridge with the nucleic acid.

The photoreactive compound of the present invention initiates a photoreaction by irradiation with light, but emphasizes the meaning that a compound that has been stable until then initiates a reaction in response to a signal of irradiation with light. Therefore, photoresponsiveness is sometimes referred to as photoresponsiveness.

（ただし、式Ｉ中、
Ｒ１１は、シアノ基、アミド基、カルボキシル基、Ｃ２～Ｃ７のアルコキシカルボニル基、又は水素原子であり、
Ｒ１２及びＲ１３は、それぞれ独立に、シアノ基、アミド基、カルボキシル基、Ｃ２～Ｃ７のアルコキシカルボニル基、又は水素原子であり、
Ｌは、リンカー部、又は単結合であり、
Ｒ２１は、アミノ基の保護基、又は水素原子である）
で表される光反応性人工アミノ酸。
（２）
Ｌが、Ｃ１～Ｃ３のアルカンジイル基、又は単結合である、（１）に記載の光反応性人工アミノ酸。
（３）
Ｒ２１が、フルオレニルメトキシカルボニル基（Ｆｍｏｃ）、ｔｅｒｔ－ブトキシカルボニル基（Ｂｏｃ）、ベンジルオキシカルボニル基（Ｃｂｚ）、及びアリルオキシカルボニル基（Ａｌｌｏｃ）からなる群から選択された保護基、又は水素原子である、（１）～（２）のいずれかに記載の光反応性人工アミノ酸。
（４）
（１）～（３）のいずれかに記載された光反応性人工アミノ酸が、ペプチド結合によって結合して含まれる、光反応性人工ポリペプチド。
（５）
核酸結合性ドメインを有するポリペプチド鎖、及び核酸結合性ドメインを有するポリペプチド鎖に対する結合性ドメインを有するポリペプチド鎖からなる群から選択されたポリペプチド鎖のアミノ酸配列中へ、
（１）～（３）のいずれかに記載された光反応性人工アミノ酸が、ペプチド結合によって導入されてなる、光反応性人工ポリペプチド。
（６）
（４）に記載された光反応性人工ポリペプチド、又は（５）に記載された光反応性人工ポリペプチドからなる、光反応性架橋剤。
（７）
ポリペプチドと核酸の間に光架橋形成する方法であって、
（４）～（５）のいずれかに記載された光反応性人工ポリペプチドと、核酸に光照射して、光反応性人工ポリペプチド中の光反応性人工アミノ酸と、核酸の塩基配列中のピリミジン塩基との間に、光架橋を形成する方法。
（８）
（１）～（３）のいずれかに記載された光反応性人工アミノ酸を、ペプチド結合によってアミノ酸配列中へ導入して、光反応性人工ポリペプチドを製造する方法。 Therefore, the present invention includes the following (1) and the following.
(1)
Equation I:

(However, in Equation I,
R11 is a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group of C2 to C7, or a hydrogen atom.
R12 and R13 are independently cyano groups, amide groups, carboxyl groups, C2-C7 alkoxycarbonyl groups, or hydrogen atoms.
L is a linker part or a single bond,
R21 is an amino protecting group or a hydrogen atom)
Photoreactive artificial amino acid represented by.
(2)
The photoreactive artificial amino acid according to (1), wherein L is an alkanediyl group of C1 to C3 or a single bond.
(3)
R21 is a protecting group selected from the group consisting of a fluorenylmethoxycarbonyl group (Fmoc), a tert-butoxycarbonyl group (Boc), a benzyloxycarbonyl group (Cbz), and an allyloxycarbonyl group (Alloc), or hydrogen. The photoreactive artificial amino acid according to any one of (1) to (2), which is an atom.
(4)
A photoreactive artificial polypeptide containing the photoreactive artificial amino acid according to any one of (1) to (3) bound by a peptide bond.
(5)
Into the amino acid sequence of a polypeptide chain selected from the group consisting of a polypeptide chain having a nucleic acid binding domain and a polypeptide chain having a binding domain to a polypeptide chain having a nucleic acid binding domain.
A photoreactive artificial polypeptide obtained by introducing the photoreactive artificial amino acid according to any one of (1) to (3) by a peptide bond.
(6)
A photoreactive cross-linking agent comprising the photoreactive artificial polypeptide according to (4) or the photoreactive artificial polypeptide according to (5).
(7)
A method of forming a photocrosslink between a polypeptide and a nucleic acid.
The photoreactive artificial polypeptide according to any one of (4) to (5) and the photoreactive artificial amino acid in the photoreactive artificial polypeptide by irradiating the nucleic acid with light, and the base sequence of the nucleic acid. A method of forming a photobridge with a pyrimidine base.
(8)
A method for producing a photoreactive artificial polypeptide by introducing the photoreactive artificial amino acid according to any one of (1) to (3) into an amino acid sequence by a peptide bond.

By using the photoreactive artificial amino acid of the present invention, a photoreactive artificial polypeptide can be obtained. The photoreactive artificial polypeptide of the present invention can form a photocrosslink with a nucleic acid by irradiation with light. The present invention provides a basic technique for forming a photocrosslink between a nucleic acid and a polypeptide.

FIG. 1 is a diagram showing a synthesis scheme (Scheme 1) of the amino acid ^CNVA A having a cyanovinylcarbazole group. FIG. 2 is a diagram showing a scheme (Scheme 2) of a photocrosslinking reaction between DNA and peptide. FIG. 3 is a gel image showing the result of examining the influence of the light irradiation time on the photocrosslinking reaction. FIG. 4A is a gel image showing the result of examining the effect of long-term light irradiation on the photocrosslinking reaction. FIG. 4B is a graph showing the effect of light irradiation time on the photocrosslinking reaction. FIG. 5 is a gel image showing the results of a photocleavage experiment of the crosslinked product. FIG. 6A is an explanatory diagram showing the positions of the bases formed by photocrosslinking in the DNA sequence, which are determined experimentally. FIG. 6B is a gel image showing the position of the photocrosslinked base in the DNA sequence. FIG. 7 is a gel image showing the results of a photocrosslinking experiment in which the chain length of the linker portion was changed.

The present invention will be described in detail below with reference to specific embodiments. The present invention is not limited to the specific embodiments listed below.

[Structure of photoreactive artificial amino acids]
The photoreactive artificial amino acid according to the present invention is represented by the following formula I:

In formula I
R11 is a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group of C2 to C7, or a hydrogen atom.
R12 and R13 are independently cyano groups, amide groups, carboxyl groups, C2-C7 alkoxycarbonyl groups, or hydrogen atoms.
L is a linker part or a single bond,
R21 is an amino protecting group or a hydrogen atom.

As the alkoxycarbonyl group, for example, C2 to C7, preferably C2 to C6, C2 to C5, C2 to C4, more preferably C2 to C3, and particularly preferably C2 can be used.

In a preferred embodiment, R11 can be a cyano group, an amide group, a carboxyl group, or an alkoxycarbonyl group of C2 to C7, and R12 and R13 can be hydrogen atoms.

In a preferred embodiment, an alkanediyl group can be used as the linker moiety for L. As the alkanediyl group, for example, C1 to C3, preferably C1 to C2 can be used, and particularly preferably a methylene group or an ethylene group.

In a preferred embodiment, L can be a methylene group, an ethylene group or a single bond. The case where L is a single bond means a state in which N and C, which are bonded to L, are bonded by a single bond.

As the protecting group for the amino group, a protecting group known as the protecting group for the amino group can be mentioned. In a preferred embodiment, the amino-protecting group is from a fluorenylmethoxycarbonyl group (Fmoc), a tert-butoxycarbonyl group (Boc), a benzyloxycarbonyl group (Cbz), and an allyloxycarbonyl group (Alloc). Protecting groups selected from the group can be used.

In a preferred embodiment, the R21 can be a fluorenylmethoxycarbonyl group (Fmoc), a tert-butoxycarbonyl group (Boc), or a hydrogen atom. When R21 is a hydrogen atom, the formula I has the structure represented by the following formula II:

[Introduction into polypeptide chain]
Since the side chain of the photoreactive artificial amino acid has a structure in which the vinylcarbazole structure represented by the above structural formula is bonded to the α carbon of the amino acid by the linker portion, the photoreactive artificial amino acid is polypolized by a peptide bond like a natural amino acid. It can be introduced into the peptide chain.

The photoreactive artificial amino acid is derived from the vinyl carbazole structure of the side chain and has photoreactivity, and the photoreaction is started by light irradiation to form a photobridge with the nucleobase. In a preferred embodiment, the nucleobase on which the vinylcarbazole structure can form photocrosslinks is a pyrimidine base located at a photocrosslinkable position. Examples of the pyrimidine base include thymine (T), cytosine (C), uracil (U), 5-methylcytosine, and 5-hydroxymethylcytosine, and thymine (T) and cytosine (C) are preferable. I can give it. This photoreactivity is maintained even when photoreactive artificial amino acids are introduced into the polypeptide chain.

[Photoresponsive artificial polypeptide]
The photoresponsive artificial polypeptide of the present invention is a polypeptide in which a photoresponsive artificial amino acid is introduced into the amino acid sequence of the polypeptide chain by a peptide bond as described above.

The introduction of the photoresponsive artificial amino acid into the polypeptide chain can be carried out by known means. That is, in a known polypeptide chain synthesis means, peptide synthesis may be carried out using a photoresponsive artificial amino acid instead of a natural amino acid or the like. Photoresponsive artificial amino acids can optionally be protected by known protecting groups for peptide synthesis. Examples of such peptide synthesis means include an Fmoc peptide solid phase synthesis method and a Boc peptide solid phase synthesis method.

The number of photoresponsive artificial amino acids introduced into the photoresponsive artificial polypeptide is not particularly limited as long as the properties required for the polypeptide chain are maintained, and may be one in the polypeptide chain. However, if desired, the number may be two or more. This can be determined as appropriate, depending on the number of pyrimidine bases desired for photocrosslinking.

[Polypeptide chain with nucleic acid binding domain]
Since the photoresponsiveness of the photoreactive artificial amino acid is maintained even when it is introduced into the polypeptide chain, the obtained polypeptide is obtained regardless of the amino acid sequence of the photoreactive artificial amino acid introduced into the polypeptide chain. Becomes a photoresponsive artificial polypeptide.

However, in a preferred embodiment, it is preferred to introduce the photoreactive artificial amino acid into the polypeptide chain having a nucleic acid binding domain. In this way, when a photoreactive artificial amino acid is introduced into a polypeptide chain having a nucleic acid-binding domain, it can be selectively bound to a nucleic acid according to the characteristics of the nucleic acid-binding domain, and can be selectively bound to a nucleic acid in the nucleic acid base sequence. The pyrimidine base at the desired position can be placed at a position where it can be photocrosslinked by the photoreactive artificial amino acid side chain. Conversely, a suitable position in the polypeptide chain into which the photoreactive artificial amino acid should be introduced can be selected depending on the position of the pyrimidine base at which photocrosslinking is desired.

Polypeptide chains having a nucleic acid binding domain also include those referred to as peptides, polypeptides or proteins. Examples of the polypeptide chain having a nucleic acid-binding domain include GCN4 (General control Noderepressible 4), SRD peptide, KEK peptide (lysine-glutamic acid-lysine peptide), and examples of the nucleic acid-binding domain. , HTH domain (helix turn helix domain), homeo domain, bZip domain (leucine zipper domain) and the like. When a polypeptide or protein having a nucleic acid-binding domain forms a dimer or a multimer, a photoreactive artificial amino acid is optionally introduced into all or part of the monomer. It can be a photoresponsive artificial polypeptide.

The nucleic acid to which the polypeptide chain having a nucleic acid-binding domain binds may be single-stranded or double-stranded, depending on its nucleic acid-binding property, and has a nucleic acid structure other than that. You may. Examples of nucleic acids include RNA and DNA.

[Peptide chain having a binding domain to a polypeptide chain having a nucleic acid binding domain]
The advantage of introducing a photoreactive artificial amino acid into a polypeptide chain having a nucleic acid binding domain is that it can bind to a nucleic acid according to the characteristics of the nucleic acid binding domain, and thus a polypeptide having a nucleic acid binding domain. Those skilled in the art understand that even a polypeptide chain that can bind to a chain can indirectly bind to a nucleic acid and can be adopted according to its characteristics. By adopting such an embodiment, for example, a photoreactive artificial polypeptide prepared by introducing a photoreactive artificial amino acid into a polypeptide chain having a binding domain to a polypeptide chain having a nucleic acid binding domain. Once prepared, it can be used in common in many types of nucleic acid-binding domains, with the advantage that it is not necessary to prepare photoreactive artificial polypeptides for each nucleic acid-binding domain.

[Photoreactive cross-linking agent]
As described above, photoreactive artificial amino acids and photoreactive artificial polypeptides can form photocrosslinks with the pyrimidine base of nucleic acid by photoreaction, and are therefore used as photoreactive crosslinkers. be able to. That is, the present invention is also in the photoreactive cross-linking agent, in the photo-reactive polypeptide-nucleic acid cross-linking agent, and in the method of forming a photo-crosslink between the polypeptide and the nucleic acid.

[Light irradiation]
Since the formation of photocrosslinks by light irradiation is a photochemical reaction, it can be carried out under a wide range of conditions such as temperature, solvent, salt concentration and pH. Therefore, it can be carried out even under physiological conditions, and can be suitably used for, for example, suppressing gene expression. In a preferred embodiment, light irradiation can be performed under the same conditions as the binding to nucleic acid by the nucleic acid binding domain. The light irradiation can be performed, for example, by irradiating light having a wavelength in the range of 340 nm to 390 nm, 360 nm to 390 nm, for example, light having a wavelength of 385 nm. Light irradiation can be performed, for example, with an irradiation time in the range of 1 minute to 300 minutes, the range of 10 minutes to 120 minutes, and the range of 30 minutes to 120 minutes. Light irradiation can be performed at a temperature in the range of, for example, 0 ° C. to 40 ° C., 0 ° C. to 30 ° C., 0 ° C. to 20 ° C., 0 ° C. to 10 ° C., for example, at a temperature under ice cooling, for example, at room temperature. Alternatively, it can be carried out, for example, at 37 ° C.

[Light cleavage]
The photocrosslink obtained by the photoreaction can be cleaved again by further irradiating with light. That is, while photocrosslinking is a covalent bond and is chemically stable, it can be cleaved by light irradiation, so that a crosslink between a polypeptide and a nucleic acid can be formed if desired, and cleaved if desired. can do. The present invention provides a widely available basic technique due to this property. The light irradiation for photo-cleavage can be performed by, for example, irradiation of light having a wavelength in the range of 300 nm to 330 nm and a wavelength in the range of 305 nm to 320 nm, for example, light having a wavelength of 312 nm.

Hereinafter, the present invention will be described in detail with reference to examples. The present invention is not limited to the examples illustrated below.

[Synthesis of ^CNVA A-containing GCN4 peptide]
Following the flow of Scheme 1 (Scheme 1), the amino acid ^CNVA A having a cyanovinylcarbazole group (Compound 7 in Scheme 1) was synthesized. FIG. 1 shows a synthesis scheme of ^CNVA A (Scheme 1).

[Synthesis of compound 2]
Carbazole (4.0 g, 23.9 mmol) is dissolved in ethanol (700 ml) at 65 ° C. After adding NaIO ₄ (1.28 g, 6.0 mmol) and I ₂ (3.02 g, 11.9 mmol) in order, add an ethanol solution (200 mL) of H ₂ SO ₄ (2.56 mL, 48.0 mmol). The reaction solution was warmed to 65 ° C. with oil bath and stirred for 2 hours. The disappearance of the raw material was confirmed by TLC (Hex: AcOEt = 4: 1), and neutralized with an ethanol solution (100 mL) of NaOH (2.24 g, 56.0 mmol). After removing ethanol, the reaction solution was extracted with chloroform (1.0 mL) and washed 3 times with water. The organic layer was dried over Na ₂ SO ₄ to remove the solvent. After purification by column chromatography (Hex: AcOEt = 4: 1) and washing with chloroform, 5 (4.47 g, 15.3 mmol, 64%) was obtained as a white powder.
^{1 1} H-NMR (400 MHz DMSO-d ₆ ): δ11.39 (s, 1H, NH), 8.49 (d, 1H, J = 1.7 Hz, H-4), 8.14 (d, 1H, J = 8.0Hz, H-5), 7,62 (dd, 1H, J = 8.4, 2.7Hz, H-2), 7.48 (d, 1H, J = 8.0Hz, H- 1), 7.40 (m, 1H, H-7), 7.33 (d, 1H, J = 8.4Hz, H-1), 7.16 (m, 1H, H-6)

[Synthesis of compound 3]
Dissolve Iodocarbazole (4.05 g, 13.8 mmol) in DMF (5.0 mL) in a microwave tube. Then, Tributylamine (3.30 mL, 13.8 mmol), Acrylonitrile (2.25 mL, 34.5 mmol) and Palladium acetate (309 mg, 1.38 mmol) are added in this order, and the reaction solution is heated at 160 ° C. at 30 ° C. using microwaves. Reacted for minutes. The disappearance of the raw material was confirmed by TLC (Hex: AcOEt = 4: 1). After removing the precipitate by Kiriyama filtration, the solvent was removed. Purification by column chromatography (CHCl ₃ ) gave 6 (2.59 g, 11.9 mmol, 86%) as a white powder.
^{1 1} H-NMR (400 MHz DMSO-d ₆ ): δ11.6 (s, 1H), 8.44 (s, 1H), 8.11 (d, 1H, J = 8.0 Hz), 7.75 (d) , 1H, J = 16.7Hz), 7.69-7.72 (m, 1H), 7.40-7.52 (m, 3H), 7.19-7.24 (m, 1H), 6 .36 (d, 1H, J = 16.7Hz)

[Synthesis of compound 4]
3-cyanovinylcarbazole (3.20 g, 14.74 mmol) and NaH (0.68 g, 28 mmol) were placed in a two-necked eggplant flask, replaced with nitrogen, and then dissolved in DMF (100 mL). After stirring for 30 minutes, dibromoacetic acid (2.56 mL, 28 mmol) was added to the reaction solution, and the mixture was stirred at 60 ° C. for 8 hours. The disappearance of the raw material was confirmed by TLC (CHCl ₃ ). After neutralizing the reaction solution with acetic acid, the precipitate was removed by Kiriyama filtration. After removing the solvent, it was dissolved in CHCl ₃ and subjected to a liquid separation operation with an aqueous NaCl solution and an aqueous NaHCO ₃ solution, and a dehydration operation was carried out using Na ₂ SO ₄ to remove the organic solvent. Then, it was purified by column chromatography (CHCl ₃ : MeOH = 99: 1) to obtain a yellow oily target product.
^{1 1} H-NMR (400 MHz DMSO-d ₆ ): δ12.22 (s, 1H), 8.34 (d, 1H, J = 8.0 Hz), 8.17 (dd, 1H, J = 4.3 Hz, 6.8Hz), 7.68 (s, 1H), 7.55-7.60 (m, 2H), 7.44 (d, 1H, 9.2Hz), 7.32-7.38 (m, 2H), 6.87 (s.1H), 5.88 (d, 1H, J = 9.2Hz)

[Synthesis of Compound 5]
To compound 4 (0.86 g, 2.4 mmol), 10 mL of a 28% aqueous ammonia solution was added on ice, and the mixture was stirred for 10 minutes and then at room temperature for 5 hours. Then, the precipitate was removed by filtration, washed twice with 1 mL of water, and dried under reduced pressure to obtain the desired product (0.30 g, 1.05 mmol) as a yellow solid in a yield of 44%.
^{1 1} H-NMR (400 MHz DMSO-d ₆ ): δ12.22 (s, 1H), 8.34 (d, 1H, J = 8.0 Hz), 8.17 (dd, 1H, J = 4.3 Hz, 6.8Hz), 7.68 (s, 1H), 7.55-7.60 (m, 2H), 7.44 (d, 1H, 9.2Hz), 7.32-7.38 (m, 2H), 6.87 (s.1H), 5.88 (d, 1H, J = 9.2Hz)

[Synthesis of Compound 6]
After dissolving cyanovinylcarbazole amino acid (620 mg, 2.1 mmol) in 0.5 mL of toluene, 1 mL of pyridine and DMAP (0.2 mg, 1.4 mmol) were added, and the mixture was stirred for 10 minutes. Then, acetic anhydride (100 μL) was slowly added dropwise. Then, it heated to 55 degreeC and stirred for 6 hours. Then, the precipitate was collected by filtration, washed with 0.5 mL of toluene and 0.5 mL of water, and dried to obtain the target product (573 mg, 1.7 mmol) as a yellow solid in a yield of 82%. ..
^{1 1} H-NMR (400 MHz DMSO-d ₆ ): δ12.24 (s, 1H), 8.36 (d, 1H, J = 8.0 Hz), 8.19 (dd, 1H, J = 4.3 Hz, 6.8Hz), 7.68 (s, 1H), 7.53-7.58 (m, 2H), 7.44 (d, 1H, J = 9.2Hz) 7.28-7.33 (m) , 2H) 5.84 (d, 1H, J = 9.2Hz) 5.74 (s, 1H)

[Synthesis of compound 7]
An acetyl protector of the cyanovinylcarbazole amino acid (600 mg, 1.8 mmol) was dissolved in 10 mL of 100 mM NaCl solution, neutralized with 1M HCl, and then 100 mg of acrylase and 10 mg cobalt hexahydrate was added. After incubating at 37 ° C. for 3 days, the precipitate was collected, washed with water and ethanol, and dried. A yellow oily target product (170 mg, 0.6 mmol) was obtained in a yield of 32%.
^{1 1} H-NMR (400 MHz DMSO-d ₆ ): δ12.22 (s, 1H), 8.34 (d, 1H, J = 8.0 Hz), 8.17 (dd, 1H, J = 4.3 Hz, 6.8Hz), 7.68 (s, 1H), 7.55-7.60 (m, 2H), 7.32-7.44 (m, 3H), 6.87 (s.1H), 5 .88 (d, 1H, J = 9.2Hz)

[Synthesis of Compound 8]
After dissolving L-cyanovinylcarbazole amino acid (300 mg, 1.03 mmol) in a mixed solvent of 1.8 mL dioxane and 0.6 mL DMF, transfer to ice and add di-tert-butyl dicarbonate (300 mg, 1.4 mmol). The mixture was stirred for 2 hours. Then, the temperature was returned to room temperature, and the mixture was further stirred for 16 hours. After participating in pH 3 using KHSO ₄ , ethyl acetate was added and an extraction operation was performed. The organic phase was dehydrated using sulfonyl ₄ to remove the organic solvent. Then, it was purified by column chromatography (CHCl ₃ : MeOH = 9: 1) to obtain an oily target product (213 mg, 0.54 mmol) in a yield of 53%.
^{1 1} H-NMR (400 MHz DMSO-d ₆ ): δ12.25 (s, 1H), 8.34 (s, 1H), 8.17 (d, 1H, J = 4.2 Hz), 7.65-7 .60 (m, 3H), 7.44 (m, 1H), 7.33 (m, 1H), 7.18 (d, 1H, J = 6.8Hz), 5.88 (d, 1H, J) = 8.2Hz), 1.42 (s, 3H)

[Peptide synthesis]
As for the synthesized amino acid, the Boc-protected body was synthesized by contract synthesis to synthesize the following sequences of PeptideA and PeptideB.
The sequences of PeptideA and PeptideB are sequences in which a part of the amino acid sequence of GCN4-bZIP basic domain (periphery interacting with nucleic acid) is taken out and one of the amino acids is replaced with a photocrosslinkable amino acid ( ^CNVA ). Is. The amino acid sequence of GCN4-bZIP basic domain is shown below.
Amino acid sequence of GCN4-bZIP basic domain:
MIVPESSDPAALKRARNTEAARRSRARKLQRMKQLEDKVEELLSKNYHLENEVARLKKLVGER

The synthesized PeptideA and PeptideB were dissolved in water, and a dimer of each peptide was prepared by the following procedure. 100 μL of 5 mM Peptide, 10 μL of 10 x PBS and 2.5 μL of 4 mM Cu (II) aqueous solution were mixed and stirred at room temperature for 1 week. Then, the dimerized peptide was separated by HPLC.
After lyophilizing the peptide dimer, the molecular weight was measured by MALDI-TOF-MS.
The sequences of PeptideA and PeptideB are summarized in Table 1. In the sequence, X represents a photocrosslinkable amino acid ( ^CNVA ).

[Photocrosslink test]
Using the above-mentioned PeptideA and PeptideB, a photocrosslinking test was performed according to the flow of Scheme 2. FIG. 2 shows a scheme (Scheme 2) of a photocrosslinking reaction between DNA and peptide. As shown in Scheme 2, the synthesized peptide was dimerized, mixed with DNA, and irradiated with light. The photocrosslinking reaction was analyzed by modified PAGE.

It has been reported that the GCN4 peptide interacts with the DNA double helix as in Scheme 2 when forming a dimer. Therefore, first, a dimer of GCN4 peptide was prepared. Copper iodide (CuI) was added to 100 μL of a 500 μM peptide aqueous solution, and an oxidation reaction was carried out at room temperature for 1 week.
In this way, the peptide and the oxidizing agent (CuI) were mixed, stirred at room temperature for 1 week, and then purified by HPLC to synthesize a heptide dimer. Then, identification by MALDI-TOF-MS was performed.
Then, 10 μM of peptide and 0.5 μM of DNA are mixed with DNA (5'-Cy3-GGATGACGTCATCC-3') in buffer (20 mM Tris-HCl, 4 mM KCl, 2 mM MgCl ₂ , 2 mM EDTA), and after annealing, 385 nm. The light irradiation was carried out at 4 ° C. Then, it was analyzed by denatured PAGE.

The test for confirming the cross-linking formation of Peptide A and Peptide B described above was carried out in detail as follows.
An optical cross-linking reaction was carried out between Peptide A and Peptide B containing ^CNV A and DNA. A 20 mM Tris-HCl (containing 4 mM KCl, 2 mM MgCl ₂ , 2 mM EDTA) aqueous solution containing 1 μM Peptide and 0.5 μM dsDNA was prepared, incubated at 37 ° C for 16 hours, further incubated at 4 ° C for 30 minutes, and then 385 nm. The light irradiation was carried out for 10 minutes. Then, after mixing 2 μL of formamide solution and 2 μL of sample, 2 μL of the mixed solution and 1 μL of 6xRoading dye were cast on a 15A acrylamide gel containing 8M urea, electrophoresed at 150V for 50 minutes, and then a gel image was obtained using LAS3000. Was acquired. The base sequence of dsDNA (double chain DNA) used is shown in Table 2. This base sequence forms double-stranded DNA by itself.
As a result of the analysis with the obtained modified PAGE, a band having a molecular weight corresponding to the crosslinked product was obtained in both Peptide A and Peptide B, that is, a photocrosslinked product was formed between the peptide A and the peptide B. I understood it.

[Examination of light irradiation time]
Regarding the cross-linking formation of Peptide A and Peptide B described above, the response to the change in the light irradiation time was examined as follows.
An optical cross-link reaction between Peptide A containing ^CNV A, Peptide B, and DNA was performed. A 20 mM Tris-HCl (containing 4 mM KCl, 2 mM MgCl ₂ , 2 mM EDTA) aqueous solution containing 1 μM Peptide and 0.5 μM dsDNA was prepared, incubated at 37 ° C for 16 hours, further incubated at 4 ° C for 30 minutes, and then 385 nm. The light irradiation was carried out for 0, 1, 5, 30 and 60 minutes. Then, after mixing 2 μL of formamide solution and 2 μL of sample, 2 μL of the mixed solution and 1 μL of 6xRoading dye were cast on a 15A acrylamide gel containing 8M urea, electrophoresed at 150V for 50 minutes, and then a gel image was obtained using LAS3000. Was acquired. The obtained image is shown in FIG. The base sequence of dsDNA (double chain DNA) used is shown in Table 2.

[Examination of light irradiation time]
Regarding the cross-linking formation of Peptide A, the response to the change in the light irradiation time over a longer period of time was examined as follows.
An optical cross-linking reaction between Peptide A and DNA was performed. The peptide sequence used and the DNA sequence are shown in Table 2 below.

A 20 mM Tris-HCl (containing 4 mM KCl, 2 mM MgCl ₂ , 2 mM EDTA) aqueous solution containing 1 μM Peptide, 0.5 μM dsDNA was prepared, incubated at 37 ° C. for 16 hours, further incubated at 4 ° C. for 30 minutes, and then 385 nm. Light irradiation was carried out for 0, 30, 60, 90, 120, 180 and 300 minutes. Then, after mixing 2 μL of formamide solution and 2 μL of sample, 2 μL of the mixed solution and 1 μL of 6xRoading dye were cast on a 15A acrylamide gel containing 8M urea, electrophoresed at 150V for 50 minutes, and then a gel image was obtained using LAS3000. Was acquired. In addition, each band was quantified from the gel image using ImageJ, and the cross-linkage ratio (%] was calculated. The results are summarized in FIG. From this result, it was found that the photocrosslinking reaction was completed with a light irradiation time (Photoradiation time) of 120 minutes.

[Photo-cleavage of crosslinked body]
The photocleavage of the crosslinked body was examined as follows.
A 20 mM Tris-HCl aqueous solution (including 4 mM KCl, 2 mM MgCl, 2 mM EDTA) containing 1 μM of the photocrosslinked body of DNA-peptide A of the sequence in Table 2 prepared above was heated at 60 ° C. using a transilluminator at 312 nm. Light irradiation was performed for 10 minutes. Then, after mixing 2 μL of formamide solution and 2 μL of sample, 2 μL of the mixed solution and 1 μL of 6x Loading dye were cast on a 15% acrylamide gel containing 25% 8M urea, and electrophoresed at 150 V for 50 minutes in a TBE buffer. After that, a gel image was acquired using LAS3000. The result is shown in FIG. In FIG. 5, the lane marked with + is the lane with light irradiation, and the lane marked with − is the control lane without light irradiation.

From this result, it was found that the photocrosslink formed between the photocrosslinkable peptide and the double-stranded DNA can be photocleaved by further light irradiation.

[Identification of crosslinked bases]
The following experiments were performed to investigate which base in the DNA the photoresponsive peptide was photocrosslinked with.
The photocrosslinked product of DNA-peptide was separated, and the DNA was cleaved by piperidine treatment or the like. The sample was lyophilized, then dissolved in 50 μL of 1 M piperidine (99.8%), heated at 90 ° C. for 30 minutes, and then the piperidine was removed under reduced pressure. Then, 10 μL of water was added, and freeze-drying was repeated multiple times to completely remove piperidine. Then, light irradiation at 60 ° C. and 312 nm was performed for 10 minutes, and then photocleavation was performed. Then, after mixing 2 μL of formamide solution and 4 μL of sample, 4 μL of the mixed solution and 1 μL of 6x Loading dye were cast on a 15A acrylamide gel containing 8M urea, electrophoresed at 300V for 30 minutes, and then gelled using LAS3000. I got the image. The results are summarized in FIGS. 6A and 6B. FIG. 6A is an explanatory diagram showing the position of the base photocrosslinked in the DNA sequence determined by this experiment. FIG. 6B is a gel image obtained by this experiment, which is a lane of a sample treated so that M1 is selectively cleaved with a pyrimidine base, and M2 is treated so as to be selectively cleaved with a thymine base. It is a sample lane, and the position in the DNA sequence of the base to be photocrosslinked was determined by comparing the positions of the bands of the M1 and M2 lanes.

As shown in FIG. 6A, X (photocrosslinkable amino acid) of Peptide A is crosslinked with C, which is the fifth base from the 3'end of DNA, and X (photocrosslinkable amino acid) of Peptide B is. , Was crosslinked with T, which is the third base from the 3'end of DNA. That is, photocrosslinkable amino acids can be photocrosslinked with thymine and cytosine (thymine and cytosine can be photocrosslinkable bases), and by changing the position of the photocrosslinkable amino acids in the peptide, they can be crosslinked in the DNA duplex. It was found that the partner base (crosslinked base) can be selected.

[Examination of chain length of linker part]
The following compound ( ^{CNV (L1)} A) in which the chain length of the linker was changed was introduced into the peptide, and how the photocrosslinking reaction between DNA and Peptide was affected by the chain length was investigated.

After preparing each peptide dimer, 10 μM peptide and 0.5 μM DNA were added to DNA (5'-Cy3-GGATGACGTCATCC-3') in buffer (20 mM Tris-HCl, 4 mM KCl, 2 mM MgCl ₂ , 2 mM EDTA). After mixing and annealing, light irradiation at 385 nm was performed at 4 ° C. Then, it was analyzed by denatured PAGE. The results are summarized in FIG. In FIG. 7, Peptide A (L1) and Peptide B (L1) include a photocrosslinkable amino acid ( ^{CNV (L1)} A) provided with a linker moiety as the photocrosslinkable amino acid of the X portion in the sequence of Table 1. It is a peptide.

From this result, in the photocrosslinkable amino acid, even when the linker portion is provided by −CH ₂ − in the connection between N of the vinylcarbazole structure and the α carbon of the amino acid structure, the same as in the case where the linker portion is not provided, as in the case where the linker portion is not provided. It was found that it can be photocrosslinked.

The present invention provides photoreactive artificial amino acids and photoreactive artificial polypeptides. The present invention is an industrially useful invention.

Claims (6)

Equation I:

(However, in Equation I,
R11 is a cyano group , a carboxyl group, an alkoxycarbonyl group of C2 to C7, or a hydrogen atom.
R12 and R13 are independently cyano groups , carboxyl groups, C2-C7 alkoxycarbonyl groups, or hydrogen atoms.
L is an alkanediyl group of C1 to C3 or a single bond.
R21 is an amino-protecting group or a hydrogen atom, and the protecting groups are fluorenylmethoxycarbonyl group (Fmoc), tert-butoxycarbonyl group (Boc), benzyloxycarbonyl group (Cbz), and allyloxycarbonyl. A protecting group selected from the group consisting of groups (Allloc) .
Photoreactive artificial amino acid represented by. Claim 1 A photoreactive artificial polypeptide containing the photoreactive artificial amino acids described in the above, which are bound by a peptide bond.And
A photoreactive artificial polypeptide in which the peptide bond is a peptide bond formed by the reaction of the amino acid structural part described below L in Formula I... Into the amino acid sequence of a polypeptide chain selected from the group consisting of a polypeptide chain having a nucleic acid binding domain and a polypeptide chain having a binding domain to a polypeptide chain having a nucleic acid binding domain.
Claim 1 A photoreactive artificial polypeptide in which the photoreactive artificial amino acid described in the above is introduced by a peptide bond.And
A photoreactive artificial polypeptide in which the peptide bond is a peptide bond formed by the reaction of the amino acid structural part described below L in Formula I... Claim 2 The photoreactive artificial polypeptide described in, orClaim 3A photoreactive cross-linking agent comprising the photoreactive artificial polypeptide described in 1. A method of forming a photocrosslink between a polypeptide and a nucleic acid.
Claims 2-3 Between the photoreactive artificial polypeptide described in any of the above and the photoreactive artificial amino acid in the photoreactive artificial polypeptide and the pyrimidine base in the base sequence of the nucleic acid by irradiating the nucleic acid with light. A method of forming a photobridge. Claim 1 A method for producing a photoreactive artificial polypeptide by introducing the photoreactive artificial amino acid described in the above into the amino acid sequence by peptide bond.And
A production method, wherein the peptide bond is a peptide bond formed by the reaction of the amino acid structural parts described below L in Formula I...

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